It is well known that genetic polymorphisms in drug metabolizing genes give rise to a variety of phenotypes. This information has been used to advantage in the past for developing genetic assays that predict phenotype and thus predict an individual's ability to metabolize a given drug. The information is of particular value in determining the likely side effects and therapeutic failures of various drugs. The availability of this sort of information will result in routine phenotyping being recommended for certain categories of patients.
Drug metabolism is carried out by the cytochrome P450 family of enzymes. For example, the cytochrome P450 isozyme gene, CYP2C9 encodes a high affinity hepatic S!-warfarin 7-hydroxylase which appears to be principally responsible for the metabolic clearance of the most potent enantiomer of warfarin. Similarly, the cytochrome P450 isozyme gene, CYP2A6, encodes a protein that metabolizes nicotine and coumarin and activates the tobacco-specific nitrosamine 4-(methyinitrosamino)-1-(3-pyridyl)-1-butanone) (NNK).
It is of note that the above gene products are also known to metabolize other substrates, for example, the CYP2C9 gene product is also known to metabolize Tolbutamide, Phenytoin, Ibuprofen, Naproxen, Tienilic acid, Diclofenac and Tetrahydrocannabinol.
It follows that genetic polymorphisms or mutations in either of the two aforementioned genes can lead to an impairment in metabolism of at least the aforementioned drugs.
In so far as CYP2C9 is concerned, sequences reported by Yasumori et al (1987 J. Biochem. 102:1075-1082.) and Kimura et al (1987 Nuc. Acids Res. 15:10053-10054) show differences at several positions including a C to T base change that results in a Arginine/Cysteine polymorphism at amino acid 144. This polymorphism has been designated R144C.
In so far as CYP2A6 is concerned, a T to A base change at position 488 of the cDNA sequence described by Yamano et al (1990 Biochemistry 29:1322-1329) results in substitution of Leucine 160 by Histidine. Henceforth this mutant form of the gene will be designated CYP2A6v1.
The variant CYP2A6v1 encodes an enzyme that is unstable and catalytically inactive. It is found in the general population at a frequency of about 1% but does not account for all slow metabolizers of coumarin.
Since the cDNA sequence structure of CYP2C9 and CYP2A6 are known, and since it is also known to perform genetic assays to determine whether a preselected mutation is present within a given gene, it should, in theory, be possible to design assays which specifically determine whether either of the aforementioned mutations are present in each of the respective aforementioned genes.
However, we have found an extraordinarily high degree of exon homology in the cytochrome P450 genes. This has resulted in non-specific binding of assay materials and poor performance of assays. In the instance where primers have been used to hybridize to genetic material, non-specific binding of such primers has taken place, and in the further instance where primers have been used to hybridize to genetic material with a view to performing a polymerase chain reactions we have found that related genes have also been amplified, for example, CYP2A7, CYP2A12 and CYP2C8 have also been amplified.